Our long range objective is to understand how HIV-1 regulates the levels of its gene products to optimize virus replication. One of the ways gene expression of HIV-1 is optimized is through alternative RNA splicing, a process which is necessary to produce the appropriate levels of the different HIV-1 mRNAs . Interference with HIV-1 alternative splicing may be a novel approach for antiviral therapy. The basic HIV-1 splicing pattern is conserved in all strains of HIV-1 suggesting its importance for viral replication. The efficiencies with which different splice sites are used are determined by the splice sites themselves and cis elements within the genome called exonic splicing silencers (ESS) and exonic splicing enhancers (ESE). The proposed studies are driven by our recent results indicating the crucial importance of some of the elements for virus replication. First, we will create additional mutations within splice sites and splicing elements and study their role in determining the level of expression of the viral Vif protein, a protein which acts to inactivate the antiviral cellular protein APOBEC3G, and in virus replication. Second, we will isolate nascent HIV-1 mRNA to determine at what point in the lifetime of the viral mRNA that splicing takes place-whether it is during or after RNA transcription is completed. Finally, we will study regulation of splicing of the outlier Group O viruses compared to the major Group M strains. Group O viruses appear to use different cis elements than Group M HIV-1 strains to regulate splicing. Identification of these elements may give us new insights about the evolution of HIV-1.